Therapeutic Dosing of a Neuregulin or a Subsequence Thereof for Treatment or Pro-phylaxis of Heart Failure

ABSTRACT

The invention relates to treatment of heart failure in a mammal. Accordingly, the invention is directed to establishing a dosing regimen whereby the therapeutic benefits conferred by administration of a neuregulin such as glial growth factor 2 (GGF2) or a subsequence thereof are maintained and/or enhanced, while concomitantly minimizing any potential side effects.

FIELD OF THE INVENTION

The field of the invention relates to treatment of heart failure. Morespecifically, the invention is directed to an improved dosing regimenwhereby the therapeutic benefits of administration of a neuregulin, suchas glial growth factor 2 (GGF2) or fragment thereof, are maintainedand/or enhanced, while minimizing any potential side effects.

BACKGROUND OF THE INVENTION

A fundamental challenge associated with the administration ofmedications to patients in need thereof is the relationship betweentolerability and efficacy. The therapeutic index is the range betweenwhich an efficacious dose of a substance can be administered to apatient and a dose at which undesired side effects to the patient arenoted. Generally, the larger the difference between the efficacious doseand the dose at which side effects initiate, the more benign thesubstance and the more likely it is to be tolerated by the patient.

Heart failure, particularly congestive heart failure (CHF), one of theleading causes of death in industrialized nations. Factors that underliecongestive heart failure include high blood pressure, ischemic heartdisease, exposure to cardiotoxic compounds such as the anthracyclineantibiotics, radiation exposure, physical trauma and genetic defectsassociated with an increased risk of heart failure. Thus, CHF oftenresults from an increased workload on the heart due to hypertension,damage to the myocardium from chronic ischemia, myocardial infarction,viral disease, chemical toxicity, radiation and other diseases such asscleroderma. These conditions result in a progressive decrease in theheart's pumping ability. Initially, the increased workload that resultsfrom high blood pressure or loss of contractile tissue inducescompensatory cardiomyocyte hypertrophy and thickening of the leftventricular wall, thereby enhancing contractility and maintainingcardiac function. Over time, however, the left ventricular chamberdilates, systolic pump function deteriorates, cardiomyocytes undergoapoptotic cell death, and myocardial function progressivelydeteriorates.

Neuregulins (NRGs) and NRG receptors comprise a growth factor-receptortyrosine kinase system for cell-cell signaling that is involved inorganogenesis and cell development in nerve, muscle, epithelia, andother tissues (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Burden etal., Neuron 18:847-855, 1997). The NRG family consists of four genesthat encode numerous ligands containing epidermal growth factor(EGF)-like, immunoglobulin (Ig), and other recognizable domains.Numerous secreted and membrane-attached isoforms function as ligands inthis signaling system. The receptors for NRG ligands are all members ofthe EGF receptor (EGFR) family, and include EGFR (or ErbB1), ErbB2,ErbB3, and ErbB4, also known as HER1 through HER4, respectively, inhumans (Meyer et al., Development 124:3575-3586, 1997; Orr-Urtreger etal., Proc. Natl. Acad. Sci. USA 90: 1867-71, 1993; Marchionni et al.,Nature 362:312-8, 1993; Chen et al., J. Comp. Neurol. 349:389-400, 1994;Corfas et al., Neuron 14:103-115, 1995; Meyer et al., Proc. Natl. Acad.Sci. USA 91:1064-1068, 1994; and Pinkas-Kramarski et al., Oncogene15:2803-2815, 1997).

The four NRG genes, NRG-1, NRG-2, NRG-3, and NRG-4, map to distinctchromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA91:9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang etal., Nature 387:509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci.USA 94:9562-9567, 1997), and collectively encode a diverse array of NRGproteins. The gene products of NRG-1, for example, comprise a group ofapproximately 15 distinct structurally-related isoforms (Lemke, Mol.Cell. Neurosci. 7:247-262, 1996 and Peles and Yarden, BioEssays15:815-824, 1993). The first-identified isoforms of NRG-1 included NeuDifferentiation Factor (NDF; Peles et al., Cell 69, 205-216, 1992 andWen et al., Cell 69, 559-572, 1992), heregulin (HRG; Holmes et al.,Science 256:1205-1210, 1992), Acetylcholine Receptor Inducing Activity(ARIA; Falls et al., Cell 72:801-815, 1993), and the glial growthfactors GGF1, GGF2, and GGF3 (Marchionni et al. Nature 362:312-8, 1993).

The NRG-2 gene was identified by homology cloning (Chang et al., Nature387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; andHigashiyama et al., J. Biochem. 122:675-680, 1997) and through genomicapproaches (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). NRG-2cDNAs are also known as Neural- and Thymus-Derived Activator of ErbBKinases (NTAK; Genbank Accession No. AB005060), Divergent of Neuregulin(Don-1), and Cerebellum-Derived Growth Factor (CDGF; PCT application WO97/09425). Experimental evidence shows that cells expressing ErbB4 orthe ErbB2/ErbB4 combination are likely to show a particularly robustresponse to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol.18:6090-6101, 1998). The NRG-3 gene product (Zhang et al., supra) isalso known to bind and activate ErbB4 receptors (Hijazi etal., Int. J.Oncol. 13:1061-1067, 1998).

An EGF-like domain is present at the core of all forms of NRGs, and isrequired for binding and activating ErbB receptors. Deduced amino acidsequences of the EGF-like domains encoded in the three genes areapproximately 30-40% identical (pairwise comparisons). Further, thereappear to be at least two sub-forms of EGF-like domains in NRG-1 andNRG-2, which may confer different bioactivities and tissue-specificpotencies.

Cellular responses to NRGs are mediated through the NRG receptortyrosine kinases EGFR, ErbB2, ErbB3, and ErbB4 of the epidermal growthfactor receptor family. High-affinity binding of all NRGs is mediatedprincipally via either ErbB3 or ErbB4. Binding of NRG ligands leads todimerization with other ErbB subunits and transactivation byphosphorylation on specific tyrosine residues. In certain experimentalsettings, nearly all combinations of ErbB receptors appear to be capableof forming dimers in response to the binding of NRG-1 isoforms. However,it appears that ErbB2 is a preferred dimerization partner that may playan important role in stabilizing the ligand-receptor complex. ErbB2 doesnot bind ligand on its own, but must be heterologously paired with oneof the other receptor subtypes. ErbB3 does possess tyrosine kinaseactivity, but is a target for phosphorylation by the other receptors.Expression of NRG-1, ErbB2, and ErbB4 is known to be necessary fortrabeculation of the ventricular myocardium during mouse development.

Neuregulins stimulate compensatory hypertrophic growth and inhibitapoptosis of myocardiocytes subjected to physiological stress. Inaccordance with these observations, administration of a neuregulin isuseful for preventing, minimizing, or reversing congestive heart diseaseresulting from underlying factors such as hypertension, ischemic heartdisease, and cardiotoxicity. See, e.g., U.S. Pat. No. 6,635,249, whichis incorporated herein in its entirety.

In view of the high prevalence of heart failure in the generalpopulation, there continues to be an unmet need to prevent or minimizeprogression of this disease, such as by inhibiting loss of cardiacfunction or by improving cardiac function

SUMMARY OF THE INVENTION

The present invention comprises a method for treating or preventingheart failure in a mammal. The method is based on the surprisingobservation that therapeutic benefits of a peptide that comprises anepidermal growth factor-like (EGF-like) domain can be achieved by dosingregimens for neuregulin administration that do not maintain steady-statesuch as by administering a therapeutically effective amount of thepeptide to a mammal at administration intervals of at or over 48, 72, 96or more hours. Accordingly, the present method calls for intermittent ordiscontinuous administration (every 48 to 96 hours, or even longerintervals) of a peptide that contains an EGF-like domain to the mammal,wherein the EGF-like domain is encoded by a neuregulin gene, and whereinadministration of the peptide is in an amount effective to treat orprevent heart failure in the mammal. Dosing regimens for neuregulinadministration that do not maintain steady-state concentrations areequally as effective as more frequent dosing regimens, yet without theinconvenience, costs or side effects that can result from more frequentadministration. As used herein the term intermittent or discontinuousadministration includes a regimen for dosing on intervals of at least 48hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, orany combination or increment thereof so long as the interval/regimen isat least 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4months. As used herein the term intermittent or discontinuousadministration includes a regimen for dosing on intervals of not lessthan 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4months, or any combination or increment thereof so long as theinterval/regimen is not less than 48 hours, 72 hours, 96 hours, 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days 1 week, 2 weeks, 4 weeks, 1 month, 2months, 3 months, 4 months.

In accordance with the present invention, intermittent or discontinuousadministration) of a peptide that contains an EGF-like domain to themammal, wherein the EGF-like domain is encoded by a neuregulin gene, isdirected to achieving a dosing regimen wherein narrow steady-stateconcentrations of the administered peptide are not maintained, therebyreducing the probability that the mammal will experience untoward sideeffects that may result from maintaining supraphysiological levels ofthe administered peptide over a prolonged duration. For example, sideeffects associated with supraphysiological levels of exogenouslyadministered NRG include nerve sheath hyperplasia, mammary hyperplasia,renal nephropathy, hypospermia, hepatic enzyme elevation, heart valvechanges and skin changes at the injection site.

In a preferred embodiment, the present invention is directed to anintermittent dosing regimen that elicts or permits fluctuations in theserum levels of the peptide comprising an EGF-like domain encoded by aneuregulin gene and thus reduces the potential for adverse side effectsassociated with more frequent administration of the peptide. Theintermittent dosing regimen of the present invention thus conferstherapeutic advantage to the mammal, but does not maintain steady statetherapeutic levels of the peptide comprising an EGF-like domain encodedby a neuregulin gene. As appreciated by those of ordinary skill in theart, there are a various embodiments of the invention to obtain theintermittent dosing; the benefits of these embodiments can be stated invarious ways for example, said administering does not maintain steadystate therapeutic levels of said peptide, the administering reducespotential for adverse side effects associated with administration of NRGpeptide more frequently, and the like.

In particular embodiments of the invention, the neuregulin may be thegene, gene product or respective subsequence or fragment thereofcomprising, consisting essentially of or consisting of: NRG-1, NRG-2,NRG-3 or NRG-4. In a preferred embodiment an NRG subsequence or fragmentof the invention comprises an epidermal growth factor-like (EGF-like)domain or a homologue thereof. As appreciated by person s of ordinaryskill in the art, a peptide homologue to an EGF-like domain peptide isdetermined by finding structural homology or by the homologue peptideperforming as a EGF-like peptide does in functional assays such as bybinding and activating ErbB receptors. Preferably the fragment is atleast 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 amino acids long. Aneuregulin peptide of the invention may, in turn, be encoded by any oneof these neuregulin genes (or subsequence thereof). In a more particularembodiment, the peptide used in the method is recombinant human GGF2 ora fragment or subsequence thereof. See FIGS. 8A-8D for the amino andnucleic acid sequences of full length human GGF2.

In an aspect of the invention, suitable mammals include, but are notlimited to, mice, rats, rabbits, dogs, monkeys or pigs. In oneembodiment of the invention, the mammal is a human.

In other embodiments of the invention, the heart failure may result fromhypertension, ischemic heart disease, exposure to a cardiotoxic compound(e.g., cocaine, alcohol, an anti-ErbB2 antibody or anti-HER antibody,such as HERCEPTIN®, or an anthracycline antibiotic, such as doxorubicinor daunomycin), myocarditis, thyroid disease, viral infection,gingivitis, drug abuse, alcohol abuse, periocarditis, atherosclerosis,vascular disease, hypertrophic cardiomyopathy, acute myocardialinfarction or previous myocardial infarction, left ventricular systolicdysfunction, coronary bypass surgery, starvation, radiation exposure, aneating disorder, or a genetic defect.

In another embodiment of the invention, an anti-ErbB2 or anti-HER2antibody, such as HERCEPTIN®, is administered to the mammal before,during, or after anthracycline administration.

In other embodiments of the invention, the peptide is administered priorto exposure to a cardiotoxic compound, during exposure to saidcardiotoxic compound, or after exposure to said cardiotoxic compound;the peptide is administered prior to or after the diagnosis ofcongestive heart failure in said mammal. A method of the invention cantake place after the subject mammal has undergone compensatory cardiachypertrophy; a method of the invention comprises that the outcome of themethod is to maintain left ventricular hypertrophy or to preventprogression of myocardial thinning, or inhibiting cardiomyocyteapoptosis. In a method of the invention, the peptide can comprising,consisting essentially of, or consisting of an EGF-like domain encodedby a neuregulin gene. A peptide of the invention is administered before,during, or after exposure to a cardiotoxic compound. In anotherembodiment, the peptide containing the EGF-like domain is administeredduring two, or all three, of these periods. In accordance with thepresent invention, the peptide containing an EGF-like domain encoded bya neuregulin gene is administered at intervals of every 48 to 96 hours.In one embodiment of the present invention, the peptide containing anEGF-like domain encoded by a neuregulin gene is GGF2. In still otherembodiments of the invention, the peptide is administered either priorto or after the diagnosis of congestive heart failure in the mammal. Inyet another embodiment of the invention, the peptide is administered toa mammal that has undergone compensatory cardiac hypertrophy. In otherparticular embodiments of the invention, administration of the peptidemaintains left ventricular hypertrophy, prevents progression ofmyocardial thinning, and/or inhibits cardiomyocyte apoptosis.

Embodiments of the invention include the following: A method fortreating heart failure in a mammal, said method comprising administeringan exogenous peptide comprising an epidermal growth factor-like(EGF-like) domain to said mammal, wherein said administering at saidintervals reduces adverse side effects associated with administration ofsaid exogenous peptide in said mammal. A method for treating heartfailure in a mammal, said method comprising administering an exogenouspeptide comprising an epidermal growth factor-like (EGF-like) domain tosaid mammal, wherein said EGF-like domain is encoded by the neuregulin(NRG)-1 gene, and said exogenous peptide is administered in atherapeutically effective amount to treat heart failure in said mammalat intervals of at least 48 hours, wherein said administering at saidintervals does not maintain steady state levels of said exogenouspeptide in said mammal. A method for treating heart failure in a mammal,said method comprising administering an exogenous peptide comprising anepidermal growth factor-like (EGF-like) domain or homologue thereof tosaid mammal, and said exogenous peptide is administered in atherapeutically effective amount to treat heart failure in said mammalat intervals of at least or not less than 48 hours, wherein saidadministering at said intervals permits intradose fluctuation of serumconcentrations of said exogenous peptide to baseline orpre-administration levels in said mammal.

As used herein, the term adverse or deleterious side effect refers to anunintended and undesirable consequence of a medical treatment. Withrespect to the present invention, an adverse or deleterious side effectresulting from administration of an exogenous peptide may include anyone or more of the following: nerve sheath hyperplasia, mammaryhyperplasia, renal nephropathy, and skin changes at the injection site.

As used herein, the term “intradose fluctuation of serum concentrationsof said exogenous peptide to pre-administration levels in said mammal”refers to the difference between serum concentration levels beforeadministration of a dose of an exogenous peptide.

As used herein, the term “steady state levels” refers to a level(s) ofan exogenous agent (e.g., a peptide) that is sufficient to achieveequilibration (within a range of fluctuation between succeeding doses)between administration and elimination. “Maintaining steady statetherapeutic levels” refers to sustaining the concentration of anexogenous agent at a level sufficient to confer therapeutic benefit to asubject or patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a histogram depicting cardiac function as exemplified bychanges in Ejection Fraction and Fractional Shortening. As indicated,rats were treated with GGF2 at 0.625 mg/kg or an equimolar amount of anEGF-like fragment (fragment; EGF-id) intravenously (iv) everyday (qday).

FIG. 2 shows a line graph depicting cardiac function as revealed bychanges in Ejection Fraction and Fractional Shortening. As indicated,rats were treated with GGF2 at 0.625 mg/kg or 3.25 mg/kg iv q day.

FIG. 3 shows a line graph depicting cardiac function as revealed bysignificant improvement in end systolic volume during the treatmentperiod. As indicated, rats were treated with GGF2 at 0.625 mg/kg or 3.25mg/kg iv q day.

FIG. 4 shows a line graph depicting cardiac function as revealed bychanges in Ejection Fraction and Fractional Shortening. As indicated,rats were treated with GGF2 3.25 mg/kg intravenously (iv) q24, 48 or 96hours.

FIG. 5 shows a line graph depicting cardiac function as revealed bychanges in the echocardiographic ejection fraction. As indicated, ratswere treated with vehicle or GGF2 3.25 mg/kg intravenously (iv), with orwithout BSA.

FIG. 6 shows a line graph depicting the half-life of recombinant humanGGF2 (rhGGF2) following iv administration.

FIG. 7 shows a line graph depicting the half-life of recombinant humanGGF2 (rhGGF2) following subcutaneous administration.

FIGS. 8A-D show the nucleic and amino acid sequences of full lengthGGF2. The nucleic acid sequence is designated SEQ ID NO: 1 and the aminoacid sequence is designated SEQ ID NO: 2.

FIG. 9 shows the nucleic and amino acid sequences of epidermal growthfactor-like (EGFL) domain 1. The nucleic acid sequence of EGFL domain 1is designated herein SEQ ID NO: 3 and the amino acid sequence of EGFLdomain I is designated herein SEQ ID NO: 4.

FIG. 10 shows the nucleic and amino acid sequences of epidermal growthfactor-like (EGFL) domain 2. The nucleic acid sequence of EGFL domain 2is designated herein SEQ ID NO: 5 and the amino acid sequence of EGFLdomain 2 is designated herein SEQ ID NO: 6.

FIG. 11 shows the nucleic and amino acid sequences of epidermal growthfactor-like (EGFL) domain 3. The nucleic acid sequence of EGFL domain 3is designated herein SEQ ID NO: 7 and the amino acid sequence of EGFLdomain 3 is designated herein SEQ ID NO: 8.

FIG. 12 shows the nucleic and amino acid sequences of epidermal growthfactor-like (EGFL) domain 4. The nucleic acid sequence of EGFL domain 4is designated herein SEQ ID NO: 9 and the amino acid sequence of EGFLdomain 4 is designated herein SEQ ID NO: 10.

FIG. 13 shows the nucleic and amino acid sequences of epidermal growthfactor-like (EGFL) domain 5. The nucleic acid sequence of EGFL domain 5is designated herein SEQ ID NO: 11 and the amino acid sequence of EGFLdomain 5 is designated herein SEQ ID NO: 12.

FIG. 14 shows the nucleic and amino acid sequences of epidermal growthfactor-like (EGFL) domain 6. The nucleic acid sequence of EGFL domain 6is designated herein SEQ ID NO: 13 and the amino acid sequence of EGFLdomain 6 is designated herein SEQ ID NO: 14.

FIG. 15 shows the amino acid sequence of a polypeptide comprising anepidermal growth factor-like (EGFL) domain, which is designated hereinSEQ ID NO: 21.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors made the surprising discovery that discontinuousor intermittent administration of a neuregulin at appropriately spacedtime intervals delivers a therapeutically effective amount of theneuregulin to a patient in need thereof and such a treatment regimen isuseful for preventing, prophylaxing, ameliorating, minimizing, treatingor reversing heart disease, such as congestive heart failure.

Despite conventional wisdom and development practice pertaining todesigning dosing regimens to maintain the most narrow range steady stateconcentrations, the present inventors demonstrate herein that dosingregimens for neuregulin administration that do not maintain narrowsteady-state concentrations are equally as effective as more frequentdosing regimens. Indeed, the present inventors have shown thatneuregulin treatment of heart failure with dosing intervals of at least48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, orany combination or increment thereof so long as the interval/regimen isat least 48 hours is as effective as daily dosing.

In order to evaluate the pharmacokinetics of exogenous NRG, the presentinventors have shown that the half-life of neuregulin when deliveredintravenously is 4 to 8 hours and when delivered subcutaneously is 11-15hours. See, e.g., Tables 1 and 2 and FIGS. 6 and 7. Dosing at regimensas infrequent as every fourth day would, therefore, not maintain anydetectable levels for at least three days between doses. Based on thesefindings, prior to the present invention, one would not have predictedthat such peak/trough ratios would correlate with consistent therapeuticbenefit. It is, noteworthy that compounds with a half-life of this orderare generally administered in accordance with a frequent dosing regimen(e.g., daily or multiple daily doses). Indeed, based on pharmacokineticdata available for GGF2, traditional development would predict thatoptimal treatment would involve daily subcutaneous dosing.

In keeping with conventional wisdom and development practice, othermedical treatments for CHF are typically administered on at least adaily basis. The periodicity of such a regimen is thought to be requiredbecause CHF is a chronic condition, commonly caused by impairedcontraction and/or relaxation of the heart, rather than an acutecondition. In persons with a weak heart leading to impaired relaxationand CHF, medical treatments include drugs that block formation or actionof specific neurohormones (e.g. angiotensin converting enzyme inhibitors(ACE-inhibitors), angiotensin receptor antagonists (ARBs), aldosteroneantagonists and beta-adrenergic receptor blockers). These and othermedications are now standard of care in chronic CHF as they have beendemonstrated to result in improved symptoms, life expectancy and/or areduction in hospitalizations. In the setting of acute exacerbation orchronic symptoms, patients are often treated with inotropes (e.g.dobutamine, digoxin) to enhance cardiac contractility, along withvasodilators (e.g. nitrates, nesiritide) and/or diuretics (e.g.furosemide) to reduce congestion. Patients with hypertension andcongestive heart failure are treated with one or more antihypertensiveagent such as beta-blockers, ACE-inhibitors and ARBs, nitrates(isosorbide dinitrate), hydralazine, and calcium channel blockers.

Thus, despite typical practice with respect to treatment of CHF, thepresent inventors have demonstrated that a novel dosing regimen resultsin effective treatment of CHF, while avoiding undesirable side-effects.Although not wishing to be bound by theory, it is likely that suchneuregulin treatment strengthens the pumping ability of the heart bystimulating cardiomyocyte hypertrophy, and partially or completelyinhibits further deterioration of the heart by suppressing cardiomyocyteapoptosis.

By way of additional background, the basic principle of dosing is todetermine an effective circulating concentration and design a dosingregimen to maintain those levels. Pharmacokinetic (PK) andpharmacodynamic (PD) studies are combined to predict a dosing regimenthat will maintain a steady-state level of a particular drug. Thetypical plan is to minimize the difference between the Cmax and Cmin andthereby reduce side-effects.

Drugs are described by their ‘therapeutic index’ which is a ratio of thetoxic dose or circulating levels divided by the effective dose orcirculating concentrations. When the therapeutic index is large there isa wide safety range where an effective dose can be given withoutapproaching toxic levels. When untoward effects result at concentrationstoo close to the effective concentrations the therapeutic index isdescribed as narrow and the drug is difficult to administer safely.

While developing dosing regimens one combines the PK/PD data withknowledge of the therapeutic index to design a dose and frequency ofadministration such that the compound is maintained at a concentrationin a patient (e.g., a human) such that it is above the effectiveconcentration and below the toxic concentration. If an effectiveconcentration of the drug cannot be maintained without inducing unsafeeffects, the drug will fail during development. Additional commentarypertaining to drug development can be found in a variety of references,including: Pharmacokinetics in Drug Development: Clinical Study Designand Analysis (2004, Peter Bonate and Danny Howard, eds.), which isincorporated herein in its entirety.

Neuregulins are growth factors related to epidermal growth factors thatbind to erbB receptors. They have been shown to improve cardiac functionin multiple models of heart failure, cardiotoxicity and ischemia. Theyhave also been shown to protect the nervous system in models of stroke,spinal cord injury, nerve agent exposure, peripheral nerve damage andchemotoxicity.

Maintaining supranormal levels of exogenously supplied neuregulins has,however, been shown to have untoward effects including nerve sheathhyperplasia, mammary hyperplasia and renal nephropathy. These effectswere observed following daily subcutaneous administration of neuregulin.See, e.g., Table 10.

As set forth herein, subcutaneous administration was explored due to theprolonged half-life compared with intravenous administration and theinitial belief that maintaining constant levels of ligand would beadvantageous. Developing dosing regimens to reduce these effects wouldsignificantly enhance the ability of neuregulins to be utilized astherapeutics and it is toward this end that the present invention isdirected. Demonstrating that less frequent dosing that does not maintainconstant levels is also effective enables this development.

Neuregulins: As indicated above, peptides encoded by the NRG-1, NRG-2,NRG-3 and NRG-4 genes possess EGF-like domains that allow them to bindto and activate ErbB receptors. Holmes et al. (Science 256:1205-1210,1992) have shown that the EGF-like domain alone is sufficient to bindand activate the p185erbB2 receptor. Accordingly, any peptide productencoded by the NRG-1, NRG-2, or NRG-3 gene, or any neuregulin-likepeptide, e.g., a peptide having an EGF-like domain encoded by aneuregulin gene or cDNA (e.g., an EGF-like domain containing the NRG-1peptide subdomains C-C/D or C-C/D′, as described in U.S. Pat. No.5,530,109, U.S. Pat. No. 5,716,930, and U.S. Pat. No. 7,037,888; or anEGF-like domain as disclosed in WO 97/09425) may be used in the methodsof the invention to prevent or treat congestive heart failure. Thecontents of each of U.S. Pat. No. 5,530,109; U.S. Pat. No. 5,716,930;U.S. Pat. No. 7,037,888; and WO 97/09425 is incorporated herein in itsentirety.

Risk Factors: Risk factors that increase the likelihood of anindividual's developing congestive heart failure are well known. Theseinclude, and are not limited to, smoking, obesity, high blood pressure,ischemic heart disease, vascular disease, coronary bypass surgery,myocardial infarction, left ventricular systolic dysfunction, exposureto cardiotoxic compounds (alcohol, drugs such as cocaine, andanthracycline antibiotics such as doxorubicin, and daunorubicin), viralinfection, pericarditis, myocarditis, gingivitis, thyroid disease,radiation exposure, genetic defects known to increase the risk of heartfailure (such as those described in Bachinski and Roberts, Cardiol.Clin. 16:603-610, 1998; Siu et al., Circulation 8:1022-1026, 1999; andArbustini et al., Heart 80:548-558, 1998), starvation, eating disorderssuch as anorexia and bulimia, family history of heart failure, andmyocardial hypertrophy.

In accordance with the present invention, neuregulins may beadministered intermittently to achieve prophylaxis such as by preventingor decreasing the rate of congestive heart disease progression in thoseidentified as being at risk. For example, neuregulin administration to apatient in early compensatory hypertrophy permits maintenance of thehypertrophic state and prevents the progression to heart failure. Inaddition, those identified to be at risk may be given cardioprotectiveneuregulin treatment prior to the development of compensatoryhypertrophy.

Neuregulin administration to cancer patients prior to and duringanthracycline chemotherapy or anthracycline/anti-ErbB2 (anti-HER2)antibody (e.g., HERCEPTIN®) combination therapy can prevent a patient'scardiomyocytes from undergoing apoptosis, thereby preserving cardiacfunction. Patients who have already suffered cardiomyocyte loss alsoderive benefit from neuregulin treatment, because the remainingmyocardial tissue responds to neuregulin exposure by displayinghypertrophic growth and increased contractility.

Therapy: Neuregulins and peptides containing EGF-like domains encoded byneuregulin genes may be administered to patients or experimental animalswith a pharmaceutically-acceptable diluent, carrier, or excipient.Compositions of the invention can be provided in unit dosage form.

Conventional pharmaceutical practice is employed to provide suitableformulations or compositions, and to administer such compositions topatients or experimental animals. Although intravenous administration ispreferred, any appropriate route of administration may be employed, forexample, parenteral, subcutaneous, intramuscular, transdermal,intracardiac, intraperitoneal, intranasal, aerosol, oral, or topical(e.g., by applying an adhesive patch carrying a formulation capable ofcrossing the dermis and entering the bloodstream) administration.

Therapeutic formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found in, forexample, “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Other potentiallyuseful parenteral delivery systems for administering molecules of theinvention include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

As a further aspect of the invention there is provided the presentcompounds for use as a pharmaceutical especially in the treatment orprevention of the aforementioned conditions and diseases. Also providedherein is the use of the present compounds in the manufacture of amedicament for the treatment or prevention of one of the aforementionedconditions and diseases.

With respect to intravenous injections, dose levels range from about0.001 mg/kg, 0.01 mg/kg to at least 10 mg/kg, in regular time intervalsof from at least about every 24, 36, 48 hours to about every 96 hoursand especially every 48, 72, or 96 hours or more as set forth herein. Ina particular embodiment, intravenous injection dose levels range fromabout 0.1 mg/kg to about 10 mg/kg, in regular time intervals of fromabout every 48 hours to about every 96 hours and especially every 48,72, or 96 hours or more as set forth herein. In another particularembodiment, intravenous injection dose levels range from about 1 mg/kgto about 10 mg/kg, in regular time intervals of from about every 48hours to about every 96 hours and especially every 48, 72, or 96 hoursor more as set forth herein. In yet another particular embodiment,intravenous injection dose levels range from about 0.01 mg/kg to about 1mg/kg, in regular time intervals of from about every 48 hours to aboutevery 96 hours and especially every 48, 72, or 96 hour or more as setforth herein s. In yet another particular embodiment, intravenousinjection dose levels range from about 0.1 mg/kg to about 1 mg/kg, inregular time intervals of from about every 48 hours to about every 96hours and especially every 48, 72, or 96 hours or more as set forthherein.

With respect to subcutaneous injections, dose levels range from about0.01 mg/kg to at least 10 mg/kg, in regular time intervals of from aboutevery 48 hours to about every 96 hours and especially every 48, 72, or96 hours or more as set forth herein. In a particular embodiment,injection dose levels range from about 0.1 mg/kg to about 10 mg/kg, inregular time intervals of from about every 48 hours to about every 96hours or more as set forth herein, and especially every 48, 72, or 96hours. In another particular embodiment, injection dose levels rangefrom about 1 mg/kg to about 10 mg/kg, in regular time intervals of fromabout every 48 hours to about every 96 hours or more as set forthherein, and especially every 48, 72, or 96 hours. In yet anotherparticular embodiment, injection dose levels range from about 0.01 mg/kgto about 1 mg/kg, in regular time intervals of from about every 48 hoursto about every 96 hours or more as set forth herein, and especiallyevery 48, 72, or 96 hours. In yet another particular embodiment,injection dose levels range from about 0.1 mg/kg to about 1 mg/kg, inregular time intervals of from about every 48 hours to about every 96hours or more as set forth herein, and especially every 48, 72, or 96hours.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses.

The compounds of the invention can be administered as the sole activeagent or they can be administered in combination with other agents,including other compounds that demonstrate the same or a similartherapeutic activity and that are determined to be safe and efficaciousfor such combined administration. Other such compounds used for thetreatment of CHF include brain natriuretic peptide (BNP), drugs thatblock formation or action of specific neurohormones (e.g. angiotensinconverting enzyme inhibitors (ACE-inhibitors), angiotensin receptorantagonists (ARBs), aldosterone antagonists and beta-adrenergic receptorblockers), inotropes (e.g. dobutamine, digoxin) to enhance cardiaccontractility, vasodilators (e.g. nitrates, nesiritide) and/or diuretics(e.g. furosemide) to reduce congestion, and one or more antihypertensiveagents such as beta-blockers, ACE-inhibitors and ARBs, nitrates(isosorbide dinitrate), hydralazine, and calcium channel blockers.

As indicated above, medical intervention involving drug treatment callsfor the selection of an appropriate drug and its delivery at an adequatedosage regimen. An adequate dosage regimen involves a sufficient dose,route, frequency, and duration of treatment. The ultimate objective ofdrug therapy is the acquisition of optimal drug concentrations at thesite of action so as to enable the treated patient to overcome thepathologic process for which treatment is necessitated. Broadlyspeaking, basic knowledge of the principles of drug dispositionfacilitates the selection of appropriate dosage regimens. Therapeuticdrug monitoring (TDM) can, however, be used in this context as asupplemental tool to assist an attending physician in determiningeffective and safe dosage regimens of selected drugs for medical therapyof individual patients.

Target Concentration and Therapeutic Window: The definition of optimaldrug concentration varies depending on the pharmacodynamic features ofthe particular drug. Optimal therapy for time-dependent antibiotics likepenicillin, for example, is related to achieving peak concentration toMIC (minimum inhibitory concentration) ratios of 2-4 and a time abovethe MIC equal to 75% of the dose interval. For concentration-dependentantibiotics like gentamicin, for example, efficacy is related toobtaining peak concentration to MIC ratios of about 8-10. Irrespectiveof the nuances associated with administration of a particular drug, drugtherapy aims to achieve target plasma concentrations (which oftenreflect the concentrations at the site of action) within the limits of a“therapeutic window”, which has been previously determined based on thepharmacokinetic, pharmacodynamic and toxicity profiles of the drug inthe target species. The width of this window varies for different drugsand species. When the difference between the minimum efficaciousconcentration and the minimum toxic concentration is small (2 to4-fold), the therapeutic window is referred to as narrow. In contrast,when there is a large difference between the effective and toxicconcentration, the drug is viewed as having a wide therapeutic window.An example of a drug with a narrow therapeutic window is digoxin, inwhich the difference between the average effective and toxicconcentrations is 2 or 3-fold. Amoxicillin, on the other hand, has awide therapeutic range and overdosing of a patient is not generallyassociated with toxicity problems.

Variability in Drug Responsiveness: Pronounced variability among healthysubjects of the same species with respect drug responsiveness is common.Moreover, disease states have the potential to affect organ systems andfunctions (e.g., kidney, liver, water content) that may in turn affectdrug responsiveness. This, in turn, contributes to increaseddifferentials in drug responsiveness in sick individuals to whom thedrug is administered. Yet another relevant issue relates toadministration of more than one drug at a time, which results inpharmacokinetic interactions that can lead to alterations inresponsiveness to one or both drugs. In summary, physiological (e.g.,age), pathological (e.g., disease effects), and pharmacological (e.g.,drug interaction) factors can alter the disposition of drugs in animals.Increased variability among individuals ensuing therefrom may result intherapeutic failure or toxicity in drugs with a narrow therapeuticindex.

The patient population that would benefit from a treatment regimen ofthe present invention is quite diverse, e.g., patients with impairedkidney function are good candidates because continuous levels of proteintherapeutics are often associated with renal glomerular deposits. Theutility of a therapeutic regimen that does not maintain constant plasmalevels as is described in this invention would, therefore, be verybeneficial for patients with compromised renal function in which anydiminution of existing function could be deleterious. Similarly, briefand intermittent exposure to a therapeutic such as GGF2, as describedherein, can be beneficial for patients with tumor types that areresponsive to chronic and continuous stimulation with a growth factor.Other patients that may specifically benefit from intermittent therapyas described herein are patients with schwannomas and other peripheralneuropathies. It is an advantage of the present invention thatintermittent dosing may have significant advantages in not maintainingcontinuous side-effect-related stimulation of various tissues.

The proper timing of blood sampling for the purposes of determiningserum drug level, as well as the interpretation of the reported levelrequire consideration of the pharmacokinetic properties of the drugbeing measured. Some terms used in discussion of these properties aredefined in the following paragraphs.

Half-Life: The time required for the serum concentration present at thebeginning of an interval to decrease by 50%. Knowing an approximatehalf-life is essential to the clinician since it determines the optimaldosing schedule with oral agents, the intradose fluctuation of the serumconcentration, and the time required to achieve steady state.

In brief, multiple pharmacokinetic studies have been performed for GGF2.Typical half-lives for GGF2 are between 4 and 8 hours for theintravenous (iv) route, whereas the half-life of subcutaneously (sc)administered GGF2 is between 11 and 15 hours. Cmax, AUC, Tmax and T½ areshown in Tables 1 and 2 below. Where the half-life was too long to bedetermined accurately by these methods a dash is presented in lieu of atime.

TABLE 1 and TABLE 2 Appendix 7 Mean Pharmacokinetics of125I-rhGGF2-Derived Radioactivity in Plasma of Male Sprague-Dawley RatsFollowing a Single intravenous or Subcutaneous Dose of 125I-rhGGF2 Group1 (n = 2) Group 2 (n = 1) Parameters Total TCAPrecip Total TCAPrecipCmax (ug eq/g) 0.3289 0.2953 0.0157 0.01 AUC 0-t (ug eq-h/g) 1.27 0.010.27 0.17 AUC inf (ug eq-h/g) 1.37 0.96 0.39 0.26 Tmax (h) 0.08 0.08 6.06.0 Half-life 6.37 6.11 13.20 14.66 Mean Pharmacokinetics of125I-rhGGF2-Derived Radioactivity in Plasma of Male Sprague-Dawley RatsFollowing a Single intravenous or Subcutaneous Dose of 125I-rhGGF2Appendix 9 Group 1 (n = 2) Group 2 (n = 1) Parameters Total TCAPrecipTotal TCAPrecip Cmax (ug eq/g) 0.2611 0.2291 0.0197 0.0034 AUC 0-t (ugeq-h/g) 1.488 0.567 0.335 0.064 AUC inf (ug eq-h/g) 1.667 0.62 — — Tmax(h) 0.08 0.08 12.0 12.0 Half-life 7.75 7.95 — — Group 1-i.v. Group2-s.c.

The plasma concentrations after administration are shown in FIGS. 6 and7 for iv and sc administration, respectively. As shown in FIGS. 6 and 7,Cmax, refers to maximal plasma concentration (the maximum concentrationthat is measured in the plasma at any time after administration);AUCinf, refers to the area under the concentration versus time curve totime infinity (which method is used to anticipate that the assay haslimits of detection); AUC0-t, refers to the area under the plasmaconcentration (time curve from time zero to the last measurableconcentration); AUC by any method refers to an estimate of the totalexposure to the animal; and Tmax, refers to the median time of maximalplasma concentration.

As is evident from the tables and figures it is not possible to maintainsteady state therapeutic levels by either dosing route with every fourthday, every other day or every day of dosing. Levels are unmeasurableafter a day and even long before that, as reflected by the data setforth in Table 11.

TABLE 11 PK Parameters for GGF2 after Intravenous Administration*AUC_(0-∞)/ AUC_(0-last)/ Dose Dose Dose AUC_(0-∞) ((hr · ng/AUC_(0-last) ((hr · ng/ CL t_(1/2) Vss (mg/kg) (hr · ng/mL) mL)/mg/kg)(hr · ng/mL) mL)/mg/kg) (mL/min/kg) (h) (mL/kg) Rats  8 16100 ± 205002010 ± 2560 16800 ± 22300 2100 ± 2790 18.1 ± 12.7  1.46 ± 1.84  1050 ±331 16 39600 ± 9440  2470 ± 590  38300 ± 10000 2390 ± 625  7.00 ± 1.33 1.69 ± 0.430  532 ± 145 Monkeys  8 15900 ± 1690  1980 ± 212  15100 ±1730  1890 ± 217  8.48 ± 0.910 2.02 ± 0.358 1110 ± 113 *taken from dataobtained from plasma GGF2 concentrations measured by ELISA. Datareported are mean ± SD.

Steady State: Steady state serum concentrations are those values thatrecur with each dose and represent a state of equilibrium between theamount of drug administered and the amount being eliminated in a giventime interval. During long term dosage with any drug, the two majordeterminants of its mean steady state serum concentration are the rateat which the drug is administered and the drug's total clearance in thatparticular patient.

Peak Serum Concentration: The point of maximum concentration on theserum concentration-versus-time curve. The exact time of the peak serumconcentration is difficult to predict since it represents complexrelationships between input and output rates.

Trough Serum Concentration: The minimum serum concentration found duringa dosing interval. Trough concentrations are theoretically present inthe period immediately preceding administration of the next dose.

Absorption: The process by which a drug enters the body. Intravascularlyadministered drugs are absorbed totally, but extravascularadministration yields varying degrees and rates of absorption. Therelationship between the rate of absorption and the rate of eliminationis the principle determinant of the drug concentration in thebloodstream.

Distribution: The dispersion of the systemically available drug from theintravascular space into extravascular fluids and tissues and thus tothe target receptor sites.

Therapeutic Range: That range of serum drug concentrations associatedwith a high degree of efficacy and a low risk of dose-related toxicity.The therapeutic range is a statistical concept: it is the concentrationrange associated with therapeutic response in the majority of patients.As a consequence, some patients exhibit a therapeutic response at serumlevels below the lower limit of the range, while others require serumlevels exceeding the upper limit for therapeutic benefit.

Correct timing of sample collection is important, since drug therapy isoften revised on the basis of serum concentration determinations. Theabsorption and distribution phases should be complete and a steady-stateconcentration achieved before the sample is drawn. Levels obtainedbefore a steady-state concentration exists may be erroneously low;increasing the dosage based on such a result could produce toxicconcentrations. In addition, when making comparative measurements, it isimportant that the sampling time be consistent.

The timing of blood samples in relation to dosage is critical forcorrect interpretation of the serum concentration result. The selectionof the time that the sample is drawn in relation to drug administrationshould be based on the phamacokinetic properties of the drug, its dosageform and the clinical reason for assaying the sample (e.g., assessmentof efficacy or clarification of possible drug-induced toxicity). Forroutine serum level monitoring of drugs with short half-lives, both asteady state peak and trough sample may be collected to characterize theserum concentration profile; for drugs with a long half-life,steady-state trough samples alone are generally sufficient.

By “congestive heart failure” is meant impaired cardiac function thatrenders the heart unable to maintain the normal blood output at rest orwith exercise, or to maintain a normal cardiac output in the setting ofnormal cardiac filling pressure. A left ventricular ejection fraction ofabout 40% or less is indicative of congestive heart failure (by way ofcomparison, an ejection fraction of about 60% percent is normal).Patients in congestive heart failure display well-known clinicalsymptoms and signs, such as tachypnea, pleural effusions, fatigue atrest or with exercise, contractile dysfunction, and edema. Congestiveheart failure is readily diagnosed by well known methods (see, e.g.,“Consensus recommendations for the management of chronic heart failure.”Am. J. Cardiol., 83(2A):1A-38-A, 1999).

Relative severity and disease progression are assessed using well knownmethods, such as physical examination, echocardiography, radionuclideimaging, invasive hemodynamic monitoring, magnetic resonanceangiography, and exercise treadmill testing coupled with oxygen uptakestudies.

By “ischemic heart disease” is meant any disorder resulting from animbalance between the myocardial need for oxygen and the adequacy of theoxygen supply. Most cases of ischemic heart disease result fromnarrowing of the coronary arteries, as occurs in atherosclerosis orother vascular disorders.

By “myocardial infarction” is meant a process by which ischemic diseaseresults in a region of the myocardium being replaced by scar tissue.

By “cardiotoxic” is meant a compound that decreases heart function bydirectly or indirectly impairing or killing cardiomyocytes.

By “hypertension” is meant blood pressure that is considered by amedical professional (e.g., a physician or a nurse) to be higher thannormal and to carry an increased risk for developing congestive heartfailure.

By “treating” is meant that administration of a neuregulin orneuregulin-like peptide slows or inhibits the progression of congestiveheart failure during the treatment, relative to the disease progressionthat would occur in the absence of treatment, in a statisticallysignificant manner. Well known indicia such as left ventricular ejectionfraction, exercise performance, and other clinical tests as enumeratedabove, as well as survival rates and hospitalization rates may be usedto assess disease progression. Whether or not a treatment slows orinhibits disease progression in a statistically significant manner maybe determined by methods that are well known in the art (see, e.g.,SOLVD Investigators, N. Engl. J. Med. 327:685-691, 1992 and Cohn et al.,N. Engl. J Med. 339:1810-1816, 1998).

By “preventing” is meant minimizing or partially or completelyinhibiting the development of congestive heart failure in a mammal atrisk for developing congestive heart failure (as defined in “Consensusrecommendations for the management of chronic heart failure.” Am. J.Cardiol., 83 (2A):1A-38-A, 1999). Determination of whether congestiveheart failure is minimized or prevented by administration of aneuregulin or neuregulin-like peptide is made by known methods, such asthose described in SOLVD Investigators, supra, and Cohn et al., supra.

The term “therapeutically effective amount” is intended to mean thatamount of a drug or pharmaceutical agent that elicits the biological ormedical response of a tissue, a system, animal or human that is beingsought by a researcher, veterinarian, medical doctor or other clinician.A therapeutic change is a change in a measured biochemicalcharacteristic in a direction expected to alleviate the disease orcondition being addressed. More particularly, a “therapeuticallyeffective amount” is an amount sufficient to decrease the symptomsassociated with a medical condition or infirmity, to normalize bodyfunctions in disease or disorders that result in impairment of specificbodily functions, or to provide improvement in one or more of theclinically measured parameters of a disease.

The term “prophylactically effective amount ” is intended to mean thatamount of a pharmaceutical drug that will prevent or reduce the risk ofoccurrence of the biological or medical event that is sought to beprevented in a tissue, a system, animal or human by a researcher,veterinarian, medical doctor or other clinician.

The term “therapeutic window” is intended to mean the range of dosebetween the minimal amount to achieve any therapeutic change, and themaximum amount which results in a response that is the responseimmediately before toxicity to the patient.

By “at risk for congestive heart failure” is meant an individual whosmokes, is obese (i.e., 20% or more over their ideal weight), has beenor will be exposed to a cardiotoxic compound (such as an anthracyclineantibiotic), or has (or had) high blood pressure, ischemic heartdisease, a myocardial infarct, a genetic defect known to increase therisk of heart failure, a family history of heart failure, myocardialhypertrophy, hypertrophic cardiomyopathy, left ventricular systolicdysfunction, coronary bypass surgery, vascular disease, atherosclerosis,alcoholism, periocarditis, a viral infection, gingivitis, or an eatingdisorder (e.g., anorexia nervosa or bulimia), or is an alcoholic orcocaine addict.

By “decreasing progression of myocardial thinning” is meant maintaininghypertrophy of ventricular cardiomyocytes such that the thickness of theventricular wall is maintained or increased.

By “inhibits myocardial apoptosis” is meant that neuregulin treatmentinhibits death of cardiomyocytes by at least 10%, more preferably by atleast 15%, still more preferably by at least 25%, even more preferablyby at least 50%, yet more preferably by at least 75%, and mostpreferably by at least 90%, compared to untreated cardiomyocytes.

By “neuregulin” or “NRG” is meant a peptide that is encoded by an NRG-1,NRG-2, or NRG-3 gene or nucleic acid (e.g., a cDNA), and binds to andactivates ErbB2, ErbB3, or ErbB4 receptors, or combinations thereof.

By “neuregulin-1,” “NRG-1,” “heregulin,” “GGF2,” or “p185erbB2 ligand”is meant a peptide that binds to the ErbB2 receptor when paired withanother receptor (ErbB1, ErbB3 or ErbB4) and is encoded by the p185erbB2ligand gene described in U.S. Pat. No. 5,530,109; U.S. Pat. No.

5,716,930; and U.S. Pat. No. 7,037,888, each of which is incorporatedherein by reference in its entirety.

By “neuregulin-like peptide” is meant a peptide that possesses anEGF-like domain encoded by a neuregulin gene, and binds to and activatesErbB2, ErbB3, ErbB4, or a combination thereof.

By “epidermal growth factor-like domain” or “EGF-like domain” is meant apeptide motif encoded by the NRG-1, NRG-2, or NRG-3 gene that binds toand activates ErbB2, ErbB3, ErbB4, or combinations thereof, and bears astructural similarity to the EGF receptor-binding domain as disclosed inHolmes et al., Science 256:1205-1210, 1992; U.S. Pat. No. 5,530,109;U.S. Pat. No. 5,716,930; U.S. Pat. No. 7,037,888; Hijazi et al., Int. J.Oncol. 13:1061-1067, 1998; Chang et al., Nature 387:509-512, 1997;Carraway et al., Nature 387:512-516, 1997; Higashiyama et al., JBiochem. 122:675-680, 1997; and WO 97/09425). See FIGS. 9-14 for nucleicand amino acid sequences corresponding to EGFL domains 1-6 encoded bythe NRG-1 gene.

By “anti-ErbB2 antibody” or “anti-HER2 antibody” is meant an antibodythat specifically binds to the extracellular domain of the ErbB2 (alsoknown as HER2 in humans) receptor and prevents the ErbB2(HER2)-dependent signal transduction initiated by neuregulin binding.

By “transformed cell” is meant a cell (or a descendent of a cell) intowhich a DNA molecule encoding a neuregulin or peptide having aneuregulin EGF-like domain has been introduced, by means of recombinantDNA techniques or known gene therapy techniques.

By “promoter” is meant a minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable based on cell type or physiological status(e.g., hypoxic versus normoxic conditions), or inducible by externalsignals or agents; such elements may be located in the 5′ or 3′ orinternal regions of the native gene.

By “operably linked” is meant that a nucleic acid encoding a peptide(e.g., a cDNA) and one or more regulatory sequences are connected insuch a way as to permit gene expression when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences.

By “expression vector” is meant a genetically engineered plasmid orvirus, derived from, for example, a bacteriophage, adenovirus,retrovirus, poxvirus, herpesvirus, or artificial chromosome, that isused to transfer a peptide (e.g., a neuregulin) coding sequence,operably linked to a promoter, into a host cell, such that the encodedpeptide or peptide is expressed within the host cell.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The discussion of documents, acts, materials, devices, articles and thelike is included in this specification solely for the purpose ofproviding a context for the present invention. It is not suggested orrepresented that any or all of these matters formed part of the priorart base or were common general knowledge in the field relevant to thepresent invention before the priority date of each claim of thisapplication.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

The following Examples will assist those skilled in the art to betterunderstand the invention and its principles and advantages. It isintended that these Examples be illustrative of the invention and notlimit the scope thereof.

EXAMPLES

As indicated herein above, the neuregulins are a family of growthfactors structurally related to Epidermal Growth Factor (EGF) and areessential for the normal development of the heart. Evidence suggeststhat neuregulins are a potential therapeutic for the treatment of heartdisease including heart failure, myocardial infarction, chemotherapeutictoxicity and viral myocarditis.

The studies described herein were served to define dosing in the leftanterior descending (LAD) artery ligation model of congestive heartfailure in the rat. Multiple neuregulin splice variants were cloned andproduced. A neuregulin fragment of consisting of the EGF-like domain(EGF-1d) from previous reports (Liu et al., 2006) was compared to afull-length neuregulin known as glial growth factor 2 (GGF2) and theEGF-like domain with the Ig domain (EGF-Ig). Male and femaleSprague-Dawley rats underwent LAD artery ligation. At 7 days postligation rats were treated intravenously (iv) with neuregulin daily.Cardiac function was monitored by echocardiography.

The first study compared 10 days of dosing with equimolar amounts ofEGF-1d or GGF2 (for GGF2 this calculates to 0.0625 and 0.325 mg/kg).GGF2 treatment resulted in significantly (p<0.05) greater improvement inEjection Fraction (EF) and Fractional Shortening (FS) than did EGF-1d atthe end of the dosing period. The second study compared 20 days of GGF2with EGF-1d and EGF-Ig at equimolar concentrations. GGF2 treatmentresulted in significantly improved EF, FS and LVESD (p<0.01).Improvements in cardiac physiology were not maintained for this periodwith either EGF-1d or EGF-1g. The third study compared daily (q 24hour), every other day (q 48 hour) and every fourth day (q 96 hour)dosing for 20 days with GGF2 (3.25 mg/kg). All three GGF2 treatmentregimens resulted in significant improvements in cardiac physiologyincluding EF, ESV and EDV and the effects were maintained for 10 daysfollowing termination of dosing. The studies presented here confirm GGF2as the lead neuregulin compound and establish optimal dosing regimensfor administering same.

As shown herein, the present studies establish the relative efficacy ofGGF2 compared with published neuregulin fragments (Liu et al., 2006),initiate dose ranging and dose frequency studies, and determine if BSAexcipient is required as previously reported.

Methods And Materials

Cloning, expression and purification of the IgEGF (Ig154Y) domain ofGGF2 (EGF-Ig) DNA: IgEGF domain was amplified from an existing GGF2 cDNAand cloned into pet 15b vector (Novagen cat #69661-3) using Ndel andBamH1 restriction sites. The resulting protein is a 21.89 kda+˜3kDa Histag (=˜25 kDa)

DNA sequence of IgEgf pet 15 clone: The underlined sequences are theprimers used for amplification. The bolded sequences are the cloningsites used to insert the sequence into the pet vector (Nde1 and BamH1).

CATATG ttgcctccccaattgaaagagatgaaaagccaggaatcggctgcaggttccaaa(SEQ ID NO: 15)       L  P  P  Q  L  K  E  M  K  S  Q  E  S  A  A  G  S  K(SEQ ID NO: 16)ctagtccttcggtgtgaaaccagttctgaatactcctctctcagattcaagtggttcaag L  V  L  R  C  E  T  S  S  E  Y  S  S  L  R  F  K  W  F  Kaatgggaatgaattgaatcgaaaaaacaaaccacaaaatatcaagatacaaaaaaagcca N  G  N  E  L  N  R  K  N  K  P  Q  N  I  K  I  Q  K  K  Pgggaagtcagaacttcgcattaacaaagcatcactggctgattctggagagtatatgtgc G  K  S  E  L  R  I  N  K  A  S  L  A  D  S  G  E  Y  M  Caaagtgatcagcaaattaggaaatgacagtgcctctgccaatatcaccatcgtggaatca K  V  I  S  K  L  G  N  D  S  A  S  A  N  I  T  I  V  E  Saacgctacatctacatccaccactgggacaagccatcttgtaaaatgtgcggagaaggag N  A  T  S  T  S  T  T  G  T  S  H  L  V  K  C  A  E  K  Eaaaactttctgtgtgaatggaggggagtgcttcatggtgaaagacctttcaaacccctcg K  T  F  C  V  N  G  G  E  C  F  M  V  K  D  L  S  N  P  Sagatacttgtgcaagtgcccaaatgagtttactggtgatcgctgccaaaactacgtaatg T  Y  L  C  K  S  P  N  E  F  T  G  D  R  C  Q  N  Y  V  M gccagcttctacGGATCC  A  S  F  Y

The final translated protein from pet15b vector is shown below. Thevector portion is underlined.

(SEQ ID NO: 17) M G G S H H H H H H G M A S M T G G T A N GV G D L Y D D D D K V P G S L P P Q L K E MK S Q E S A A G S K L V L R C E T S S E Y SK I Q K K P G K S E L R I N K A S L A D S GE Y M C K V I S K L E N D S A S A N I T I VE S N A T S T S T T G T S H L V K C A E K EK T F C V N G G E C F M V K D L S N P S R YL C K C P N E F T G D R C Q N Y V M A S F Y

Protein expression: The clone was transformed into B121 cells forprotein expression using the Overnight Express Autoinduction System(Novagen) in LB media at 25° C. for 24 hours.

Protein Refolding: Adapted from Novagen Protein Refolding Kit, 70123-3.Protein Purification: His TRAP columns—as per manufacturer'sinstructions

Western blotting: Protein expression was assessed by western blotting.Resulting band with the His tag runs at around 25 kD.

A 4-20% criterion gel (Biorad) was used for protein resolution followedby transfer onto Protran nitrocellulose paper (0.1 μm pore size fromSchliecher and Schull). The blot is blocked in 5% milk in TBS-T (0.1%).Primary antibody (Anti EGF Human NRG1-alpha/HRG1-alpha Affinity PurifiedPolyclonal Ab Cat # AF-296-NA from R&D systems) 1:1000 dilution in 5%milk in TBS-T—1 hour at RT (also works at 4° C. overnight). Rabbit antigoat HRP secondary antibody was used at 1:10,000 dilution in 5% milk inTBS-T for 1 hour at RT. All washes were performed in TBS-T

Purification Protocol for Ig154Y: The cultures are grown at 25° C. inOvernight Express Autoinduction System 1 from Novagen (cat #71300-4).The culture is spun down and the pellets are extracted, solubilized andre-folded to acquire the Ig154Y before purification can take place.

Materials For Extraction, Solubilization And Re-Folding

10× Wash Buffer: 200 mM Tris-HCl, pH 7.5, 100 mM EDTA, 10% Triton X-100

10× Solubilization Buffer: 500 mM CAPS, pH 11.0

50× Dialysis Buffer: 1M Tris-HCl, pH 8.5

30% N-laurylsarcosine—add as powder (Sigma 61739-5G)

1M DTT

Reduced glutathione (Novagen 3541)

Oxidized glutathione (Novagen 3542)

A. Cell Lysis And Preparation of Inclusion Bodies

-   -   Cell pellets were thawed and re-suspended in 30 mls 1× wash        buffer.    -   Protease inhibitors (25 ul of 10× per 50 mls), DNase (200 ul of        1 mg/ml per 50m1) and MgCl₂ (500 ul of 1M per 50 mls) were added        to suspension.    -   Cells were lysed by sonication with cooling on ice.    -   Following sonication inclusion bodies were collected by        centrifugation at 10000×g for 12 minutes.    -   Supernatant was removed and the pellet thoroughly re-suspended        in 30 mls of 1× Wash Buffer.    -   Step 4 was repeated.    -   The pellet was thoroughly re-suspended in 30 mls of 1× Wash        Buffer.    -   The inclusion bodies were collected by centrifugation at 10000×g        for 10 minutes.

B. Solubilization And Refolding

-   -   From the wet weight of inclusion bodies to be processed,        calculate the amount of 1× Solubilization Buffer necessary to        re-suspend the inclusion bodies at a concentration of 10-15        mg/ml. If the calculated volume is greater than 250 ml, use 250        ml.    -   At room temperature, prepare the calculated volume of 1×        Solubilization Buffer supplemented with 0.3% N-laurylsarcosine        (up to 2% may be used if needed in further optimization) (300        mg/100 mL buffer) and 1 mM DTT.    -   Add the calculated amount of 1× Solubilization Buffer from step        2 to the inclusion bodies and gently mix. Large debris can be        broken up by repeated pipetting.    -   Incubate in refrigerator shaker at 25° C., 50-100 rpm for 4-5        hours (or longer if needed in further optimization).    -   Clarify by centrifugation at 10000×g for 10 minutes at room        temperature    -   Transfer the supernatant containing the soluble protein into a        clean tube.

C. Dialysis Protocol For Protein Refolding

-   -   Prepare the required volume of buffer for dialysis of        solubilized protein. The dialysis should be performed with at        least 2 buffer changes of greater than 50 times the volume of        the sample. Dilute the 50× Dialysis Buffer to 1× at the desired        volume and supplement with 0.1 mM DTT.    -   Dialyze for at least 4 hours at 4° C. Change the buffer and        continue. Dialyze for an additional 4 or more hours.    -   Prepare additional dialysis buffer as determined in step 1, but        omit DTT.    -   Continue the dialysis through two additional changes (minutes 4        hr each), with the dialysis buffer lacking DTT.

D. Redox Refolding Buffer To Promote Disulfide Bond Formation

-   -   Prepare a dialysis buffer containing 1 mM reduced glutathione        (1.2 g/4 L) and 0.2 mM oxidized glutathione (0.48 g/4 L) in 1×        Dialysis Buffer. The volume should be 25 times greater than the        volume of the solubilized protein sample. Chill to 4° C.    -   Dialyze the refolded protein from step 1 overnight at 4° C.

Materials For Purification

All procedures are done at 4° C.

Chemicals:

Trizma Hydrochloride (Sigma T5941-500G)

Sodium Chloride 5M Solution (Sigma 56546-4L)

Sodium Hydroxide 10N (JT Baker 5674-02)

Imidazole (JT Baker N811-06)

A. Purification on the HISPrep FF 16/10 Column—20 mls (GE Healthcare)

Buffer A: 20 mM Tris-HCL+500 mM NaCl pH 7.5

Buffer B: Buffer A+500 mM Imidazole pH 7.5

Equilibration of column: Buffer A—5 CV, Buffer B—5 CV, Buffer A—10 CV

Load 20 ml of sample per run on 20 ml column at 0.5 ml/min

Wash column with 5 CV of buffer A

Elute column with 5 CV of 280 mM Imidazole.

Clean with 10 CV of 100% Buffer B.

Equilibrate with 15 CV of Buffer A

Analyze fractions with a SDS-page silver stain

Pool fractions with Ig154Y

B. His-Tag Removal

Removal of the His-Tag is done with A Thrombin Cleavage Capture Kit fromNovagen (Cat #69022-3). Based on previous testing, the best conditionsare room temperature for 4 hours with Thrombin at 0.005 U of enzyme perμl for every 10 μg of Ig154Y protein. After four hours of incubation,add 16 μl of Streptavidin Agarose slurry per unit of Thrombin enzyme.Rock sample for 30 minutes at room temp. Recover the Ig154Y throughspin-filtration or sterile filtering (depending on volume).

Full cleavage is determined by EGF and Anti-His western blotting.

C. Concentration of Ig154Y

Adjust to desired concentration with Millipore Centriprep 3000 MWCO 15ml concentrator (Ultracel YM-3, 4320)

D. Storage In Final Buffer

Store in 20 mM Tris+500 mM NaCl pH 7.5 and 1× PBS+0.2% BSA.

Cloning, expression and purification of 156Q (EGF-Id) [NRG1b2 EGF domain(156Q)] DNA: NRG1b2 egf domain was cloned from human brain cDNA andcloned into pet 15b vector (Novagen cat #69661-3) using Nde1 and BamH1restriction sites. The resulting protein is a 6.92 kda+˜3 kDa His tag(=9.35 kDa)

DNA Sequence of NRG1b2 egf pet 15 Clone

The underlined sequences are the cloning sites (Nde1 and BamH1)

(SEQ ID NO: 18) CATATGAGCCA TCTTGTAAAA TGTGCGGAGA AGGAGAAAACTTTCTGTGTG AATGGAGGGG AGTGCTTCAT GGTGAAAGACCTTTCAAACC CCTCGAGATA CTTGTGCAAG TGCCCAAATGAGTTTACTGG TGATCGCTGC CAAAACTACG TAATGGCCAGCTTCTACAAG GCGGAGGAGC TGTACCAGTA AGGATCC

The final translated protein from pet15b vector is shown below. The egfdomain is highlighted in green.

(SEQ ID NO: 19)                   10                20MGSSHHHHHH SSGLVPRGSH MSHLVKCAEK EKTFCVNGGE         30             60              70       80CFMVKDLSNP SRYLCKCPNE FTGDRCQNYV MASFYKAEEL YQ 

Calculated pI/Mw: 7.69/9349.58

Protein Expression

The clone was transformed into B121 cells for protein expression usingthe Overnight Express Autoinduction System (Novagen)) in LB media at 25°C. for 24 hours. Expression is primarily in insoluble inclusion bodies.

Protein Refolding: Adapted from Novagen Protein Refolding Kit, 70123-3.

Protein Purification: Protein is loaded onto an anion exchange columnDEAE at 2.5 ml/min. The EGF-Id fragment remains in the flow through,whereas the contaminants bind and elute at a higher salt. The loadingand washing buffer is 50 mM Tris pH7.9 and elution buffer is 50 mM TrispH7.9 with 1M NaCl. The flow through is pooled and concentrated withCentriprep YM-3 from Millipore.

Western blotting: Protein expression is assessed by western blotting.Resulting band runs at around 10 kD.

A 4-20% criterion gel (Biorad) was used for protein resolution followedby transfer onto Protran nitrocellulose paper (0.1 μm pore size fromSchliecher and Schull). The blot is blocked in 5% milk in TBS-T (0.1%).Primary antibody (Anti EGF Human NRG1-alpha/HRG1-alpha Affinity PurifiedPolyclonal Ab Cat #AF-296-NA from R&D systems) 1:1000 dilution in 5%milk in TBS-T—1 hour at RT (also works at 4° C. overnight). Rabbit antigoat HRP secondary antibody was used at 1:10,000 dilution in 5% milk inTBS-T for 1 hour at RT. All washes were performed in TBS-T

Purification Protocol For NRG-156Q

The cultures are grown at 25° C. in Overnight Express AutoinductionSystem 1 from Novagen (cat #71300-4). There is very little solubleNRG-156Q (EGF-Id) present. The culture is spun down and the pellets areextracted, solubilized and re-folded to acquire the NRG-156Q beforepurification can take place.

Materials For Extraction, Solubilization And Re-Folding

10× Wash Buffer: 200 mM Tris-HCl, pH 7.5, 100 mM EDTA, 10% Triton X-100

10× Solubilization Buffer: 500 mM CAPS, pH 11.0

50× Dialysis Buffer: 1M Tris-HCl, pH 8.5

30% N-laurylsarcosine—add as powder (Sigma 61739-5G)

1M DTT

Reduced glutathione (Novagen 3541)

Oxidized glutathione (Novagen 3542)

A. Cell Lysis And Preparation of Inclusion Bodies

-   -   Thaw and re-suspend cell pellet in 30 mls 1× wash buffer. Mix as        needed for full re-suspension.    -   Add protease inhibitors (25 ul of 10× per 50 mls), DNase (200 ul        of 1 mg/ml per 50 ml) and MgCl2 (500 ul of 1M per 50 mls) to        suspension.    -   Lyse the cells by sonication.        -   a. Cool the cells on ice throughout this step.        -   b. Using the square tip, sonicate for 30 seconds on level 6,            10 times until suspension becomes less viscous. Let            suspension cool on ice for 60 seconds between each            sonication. Keep volume no higher than 40 mls in 50 ml            conical tube when sonicating.    -   When complete, transfer each suspension to 250 ml angled neck        centrifuge bottles for use with F-16/250 rotor.    -   Collect the inclusion bodies by centrifugation at 10,000×g for        12 minutes.    -   Remove the supernatant (save a sample for analysis of soluble        protein) and thoroughly re-suspend the pellet in 30 mls of 1×        Wash Buffer.    -   Repeat centrifugation as in Step 4 and save the pellet.    -   Again, thoroughly re-suspend the pellet in 30 mls of IX Wash        Buffer.    -   Collect the inclusion bodies by centrifugation at 10,000×g for        10 minutes. Decant the supernatant and remove the last traces of        liquid by tapping the inverted tube on a paper towel.

B. Solubilization And Refolding

-   -   From the wet weight of inclusion bodies to be processed,        calculate the amount of 1× Solubilization Buffer necessary to        re-suspend the inclusion bodies at a concentration of 10-15        mg/ml. If the calculated volume is greater than 250 ml, use 250        ml.    -   At room temperature, prepare the calculated volume of 1×        Solubilization Buffer supplemented with 0.3% N-laurylsarcosine        (up to 2% may be used if needed in further optimization) (300        mg/100 mL buffer) and 1 mM DTT.    -   Add the calculated amount of 1× Solubilization Buffer from step        2 to the inclusion bodies and gently mix. Large debris can be        broken up by repeated pipetting.    -   Incubate in refrigerator shaker at 25° C., 50-100 rpm for 4-5        hours.    -   Clarify by centrifugation at 10,000×g for 10 minutes at room        temperature.

C. Dialysis Protocol For Protein Refolding

-   -   Prepare the required volume of buffer for dialysis of        solubilized protein. The dialysis should be performed with at        least 2 buffer changes of greater than 50 times the volume of        the sample.    -   Dilute the 50× Dialysis Buffer to 1× at the desired volume and        supplement with 0.1 mM DTT.    -   Dialyze for at least 4 hours at 4° C. Change the buffer and        continue. Dialyze for an additional 4 or more hours.    -   Prepare additional dialysis buffer as determined in step 1, but        omit DTT.    -   Continue the dialysis through two additional changes (minutes 4        hours each), with the dialysis buffer lacking DTT.

D. Redox Refolding Buffer To Promote Disulfide Bond Formation

-   -   Prepare a dialysis buffer containing 1 mM reduced glutathione        (1.2 g/4 L) and 0.2 mM oxidized glutathione (0.48 g/4 L) in 1×        Dialysis Buffer. The volume should be 25 times greater than the        volume of the solubilized protein sample. Chill to 4° C.    -   Dialyze the refolded protein from step 1 overnight at 4° C.

Materials For Purification

All procedures are done at 4° C.

Chemicals:

Trizma Hydrochloride (Sigma T5941-500G)

Sodium Chloride 5M Solution (Sigma 56546-4L)

Sodium Hydroxide ION (JT Baker 5674-02)

E. Purification On the DEAE HiPrep 16/10 Anion Column—20 mls (GEHealthcare)

Buffer A: 50 mM Tris-HCL pH 8.0

Buffer B: 50 mM Tris-HCL with 1M NaCl pH 8.0

Equilibration of column: Buffer A—5 CV, Buffer B—5 CV, Buffer A—10 CV

-   -   Load 50 ml of sample per run on 20 ml column at 2.0 ml/min        (NRG-156 (EGF-Id) is in the flow through).    -   Wash 20 ml column with 5 CV of buffer A

20 ml column with gradient to 100% B with 5 CV. This is to elute offcontaminants.

-   -   Clean with 10 CV of 100% Buffer B.    -   Equilibrate with 15 CV of Buffer A    -   Analyze fractions with a SDS-page silver stain    -   Pool fractions with NRG-156Q (lOkDa)

F. Concentration of NRG-156 (EGF-Id)

-   -   Concentrate with Millipore Centriprep 3000 MWCO 15 ml        concentrator (Ultracel YM-3, 4320)    -   Use Modified Lowry Protein Assay to determine concentration.

G. His-Tag Removal

Removal of the His-Tag is done with A Thrombin Cleavage Capture Kit fromNovagen (Cat #69022-3). Based on previous testing the best conditionsare room temperature for 4 hours with Thrombin at 0.005 U of enzyme perμl for every 10 μg of NRG-156Q (EGF-Id) protein. After four hours ofincubation, add 16 μl of Streptavidin Agarose slurry per unit ofThrombin enzyme. Rock sample for 30 minutes at room temperature. Recoverthe NRG-156Q through spin-filtration or sterile filtering (depending onvolume). Complete cleavage is determined with an EGF and Anti-Hiswestern.

H. Storage In Final Buffer

Stored in 1× PBS with 0.2% BSA at 4° C.

Expression And Purification of GGF2

For the cloning and background information for GGF2, see U.S. Pat. No.5,530,109. The cell line is described in U.S. Pat. No. 6,051,401. Theentire contents of each of U.S. Pat. No. 5,530,109 and U.S. Pat. No.6,051,401 is incorporated herein by reference in its entirety.

CHO-(Alpha2HSG)-GGF cell line: This cell line was designed to producesufficient quantities of fetuin (human alpha2HSG) to support highproduction rates of rhGGF2 in serum free conditions.

Cho (dhfr−) cells were transfected with the expression vector shownbelow (pSV-AHSG). Stable cells were grown under ampicillin selection.The cell line was designated (dhff⁻/α2HSGP). The dhfr⁻/α2HSGP cells werethen transfected with the pCMGGF2 vector shown below containing thecoding sequence for human GGF2 using the cationic lipid DMRIE-C reagent(Life Technologies #10459-014).

Stable and high producing cell lines were derived under standardprotocols using methotrexate (100 nM, 200 nM, 400 nM, 1 μM) at 4-6 weeksintervals. The cells were gradually weaned from serum containing media.Clones were isolated by standard limiting dilution methodologies.Details of the media requirements are found in the above mentionedreports.

To enhance transcription, the GGF2 coding sequence was placed after theEBV BMLF-1 intervening sequence (MIS). See diagrams below.

MIS Sequence (SEQ ID NO: 20)CGAT[AACTAGCAGCATTTCCTCCAACGAGGATCCCGCAG(GTAAGAAGCTACACCGGCCAGTGGCCGGGGCCCGATAACTAGCAGCATTTCCTCCAACGAGGATCCCGCAG(GTAAGAAGCTACACCGGCCAGTGGCCGGGGCCGTGGAGCCGGGGGCATCCGGTGCCTGAGACAGAGGTGCTCAAGGCAGTCTCCACCTTTTGTCTCCCCTCTGCAG)AGAGCCACATTCTGGAA]GTT                       atgagatgg cgacgcgccc cgcgccgctc cgggcgtccc 301ggcccccggg cccagcgccc cggctccgcc gcccgctcgt cgccgccgct gccgctgctg 361ccactactgc tgctgctggg gaccgcggcc ctggcgccgg gggcggcggc cggcaacgag 421gcggctcccg cgggggcctc ggtgtgctac tcgtccccgc ccagcgtggg atcggtgcag 481gagctagctc agcgcgccgc ggtggtgatc gagggaaagg tgcacccgca gcggcggcag 541cagggggcac tcgacaggaa ggcggcggcg gcggcgggcg aggcaggggc gtggggcggc 601gatcgcgagc cgccagccgc gggcccacgg gcgctggggc cgcccgccga ggagccgctg 661ctcgccgcca acgggaccgt gccctcttgg cccaccgccc cggtgcccag cgccggcgag 721cccggggagg aggcgcccta tctggtgaag gtgcaccagg tgtgggcggt gaaagccggg 781ggcttgaaga aggactcgct gctcaccgtg cgcctgggga cctggggcca ccccgccttc 841ccctcctgcg ggaggctcaa ggaggacagc aggtacatct tcttcatgga gcccgacgcc 901aacagcacca gccgcgcgcc ggccgccttc cgagcctctt tcccccctct ggagacgggc 961cggaacctca agaaggaggt cagccgggtg ctgtgcaagc ggtgcgcctt gcctccccaa 1021ttgaaagaga tgaaaagcca ggaatcggct gcaggttcca aactagtcct tcggtgtgaa 1081accagttctg aatactcctc tctcagattc aagtggttca agaatgggaa tgaattgaat 1141cgaaaaaaca aaccacaaaa tatcaagata caaaaaaagc cagggaagtc agaacttcgc 1201attaacaaag catcactggc tgattctgga gagtatatgt gcaaagtgat cagcaaatta 1261ggaaatgaca gtgcctctgc caatatcacc atcgtggaat caaacgctac atctacatcc 1321accactggga caagccatct tgtaaaatgt gcggagaagg agaaaacttt ctgtgtgaat 1381ggaggggagt gcttcatggt gaaagacctt tcaaacccct cgagatactt gtgcaagtgc 1441ccaaatgagt ttactggtga tcgctgccaa aactacgtaa tggccagctt ctacagtacg 1501tccactccct ttctgtctct gcctgaatag GGF2 Protein Sequence (SEQ ID NO: 2)-MRWRRAPRRSGRPGPRAQRPGSAARSSPPLPLLPLLLLLGTAALAPGAAAGNEAAPAGASVCYSSPPSVGSVQELAQRAAVVIEGKVHPQRRQQGALDRKAAAAAGEAGAWGGDREPPAAGPRALGPPAEEPLLAANGTVPSWPTAPVPSAGEPGEEAPYLVKVHQVWAVKAGGLKKSDLLTVRLGTWGHPAFPSCGRLKEDSRYIFFMEPDANSTSRAPAAFRASFPPLETGRNLKKEVSRVLCKRCALPPQLKEMKSQESAAGSKLVLRCETSSEYSSLRKFWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNATSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYSTSTPFLSLPE

GGF2 production: One vial of GGF2 at 2.2×10⁶ cells/mL was thawed into100 mls of Acorda Medium 1 (see Table 3) and expanded until reachingsufficient numbers to seed production vessels. Cells were inoculatedinto the production media Acorda Medium 2 (see Table 4) at 1.0×10⁵cells/mL in two liter vented roller bottles. Roller bottles aremaintained at 37° C. for 5 days and then reduced to 27° C. for 26 days.The roller bottles are monitored for cell count and general appearancebut they are not fed. Once viability is below 10% the cells are spun outand conditioned media harvested and sterile filtered.

TABLE 3 Medium 1 Catalog Final Item Vendor Number concentration CD-CHOInvitrogen 10743-029 remove 50 ml, then add components below FeSO₄•EDTASigma F-0518 1x (10 ml/L) L-Glutamine Cellgro 25-005-Cl 4 mM (20 ml/L)Recombinant Sigma I-9278 290 U/L (1 ml/L) Human Insulin Non-essentialCellgro 25-025-Cl 1x (10 ml/L) amino acid Peptone Type 4 Sigma P0521Powder-Made 20X in Soybean-HySoy CD-CHO (50 ml/L) Gentamicin Invitrogen15750-078 100 μg (2 ml/L)

TABLE 4 Medium 2 Catalog Final Item Vendor Number concentration CD-CHOInvitrogen 10743-029 50% (−50 ml first) HyQ SFX-CHO HyClone SH30187.0250% (−50 ml first) FeSO₄•EDTA Sigma F-0518 1x (10 ml/L) L-GlutamineCellgro 25-005-Cl 4 mM (20 ml/L) Recombinant Sigma I-9278 290 U/L (1ml/L) Human Insulin Non-essential Cellgro 25-025-Cl 1x (10 ml/L) aminoacid Peptone Type 4 Sigma P0521 Powder-Made 20X in Soybean-HySoy CD-CHO(50 ml/L) Gentamicin Invitrogen 15750-078 100 μg (2 ml/L)

Purification Protocol For GGF2

All procedures are done at 4° C.

Chemicals:

-   -   Sodium Acetate    -   Glacial Acetic Acid (for pH adjustment)    -   10N NaOH (for pH adjustment)    -   NaCl    -   Sodium Sulfate    -   L-Arginine (JT Baker cat #: 2066-06)    -   Mannitol (JT Baker cat #: 2553-01)

Starting material: Conditioned media supernatant. Adjust pH to 6.5.

Step1

Capture—Cation Exchange Chropmatography

-   -   HiPrep SP 16/10 (Amersham Biosciences)    -   Column equilibration: Buffer A—5 CV, buffer B—5 CV, buffer 15%        B—5 CV    -   Buffer A: 20 mM NaAcetate, pH 6.0    -   Buffer B: 20 mM NaAcetate, pH 6.0, 1M NaCl

Load sample at 2 ml/min with a continuous load overnight if possible.Binding is better with continuous loading.

-   -   Maximum capacity for a starting sample: 5 mg GGF2/ml media    -   Flow rate: 3 ml/min    -   First wash: 15% B, 10 CV    -   Second wash: 35% B, 10 CV    -   GGF2 elution: 60% B, 8 CV    -   Column wash: 100% B, 8 CV

Buffers: Composition Conductivity Use  15% B 20 mM NaAcetate,Preequilibration pH 6.0, 150 mM NaCl First wash  35% B 20 mM NaAcetate,Second wash pH 6.0, 350 mM NaCl  60% B 20 mM NaAcetate, GGF2 elution pH6.0, 600 mM NaCl 100% B 20 mM NaAcetate, 88 mS/cm Column wash pH 6.0,1000 mM NaCl

Step 2

Refinement—Gel Filtration Chromatography

-   -   Sephacryl 5200 26/60    -   Elution buffer: 20 mM NaAcetate, 100 mM Sodium Sulfate, 1%        mannitol, 10 mM L-Arginine, pH 6.5    -   Buffer conductivity:    -   Sample: SP GGF2 elution pool concentrated up to ˜AU280 1.0    -   Flow rate: 1.3 ml/min    -   Peak elution: at ˜0.36 CV from injection start

Step 3

DNA And Endotoxin Removal—Filtration Through Intercept Q Membrane

-   -   Preequilibration buffer: 20 mM NaAcetate, 100 mM Sodium Sulfate,        1% Mannitol, 10 mM L-Arginine, pH 6.5    -   Collect flow through

Step 4

Final Formulation And Sample Preparation

-   -   Add additional 90 mM L-Arginine to the sample    -   Concentrate    -   Sterile Filter

The vehicle/control article used herein is 0.2% Bovine Serum Albumin(BSA), 0.1 M Sodium Phosphate, pH 7.6.

Rat strains CD®IGS [Cr1:CD®SD)/MYOINFARCT] and Naive Sprague Dawley areused herein. These strains were acquired from Charles RiverLaboratories. The test animals are approximately 6-7 weeks of age atarrival and weigh approximately 160-200 g, at the time of surgicalprocedure. The actual range may vary and is documented in the data.

All naive Sprague Dawley animals received were placed on study andassigned to Group 1. Animals considered suitable for study were weighedprior to treatment.

All CD®IGS [Cr1:CD®(SD)/MYOINFARCT] animals received were randomizedinto treatment groups (Groups 2-5) using a simple randomizationprocedure based on calculated Ejection Fraction from Echocardiographicexaminations performed on Day 7 post surgical procedure conducted atCharles River Laboratories. Simple randomization was conducted to resultin each treatment group (Groups 2-5) consisting of applicable numbers ofanimals resulting in an approximately equal Group Mean Ejection Fraction(±3%) across Group 2-5.

All animals in Group 2-6 were acclimated at Charles River Laboratoriesaccording to Standard Operating Procedures of that laboratory. Animalswere subsequently randomized into treatment groups. All naive animals inGroup 1 were acclimated for approximately 24 hours post receipt prior totheir primary Echocardiographic examinations.

The animals were individually housed in suspended, stainless steel,wire-mesh type cages, Solid-bottom cages were not used in generalbecause rodents are coprophagic and the ingestion of feces containingexcreted test article and metabolic products or ingestion of the beddingitself could confound the interpretation of the results in this toxicitystudy.

Fluorescent lighting was provided via an automatic timer forapproximately 12 hours per day. On occasion, the dark cycle wasinterrupted intermittently due to study-related activities. Temperatureand humidity were monitored and recorded daily and maintained to themaximum extent possible between 64 to 79° F. and 30 to 70%,respectively.

The basal diet was block Lab Diet® Certified Rodent Diet #5002, PMINutrition International, Inc. This diet was available ad libitum unlessdesignated otherwise. Each lot number used was identified in the studyrecords. Tap water was supplied ad libitum to all animals via anautomatic water system unless otherwise indicated.

STUDY DESIGNS

TABLE 5 GGF2 versus EGF-ld fragment (Liu et al., 2006) Dosed for 10 daysstarting day 7 after LAD ECHO Time In-Life Dosing Points Group TreatmentDuration Dose Interval† (post-op) 1 Control 17 days Vehicle 24 Hr Day 6,17 (n = 5 M; n = 5 F) (Vehicle) post-op only 2 17 days 0.0625 24 Hr Day6, 17 (n = 6 M; n = 6 F) GGF2 post mg/kg 3 GGF2 17 days 0.625 24 Hr Day6, 17 (n = 6 M; n = 6 F) post mg/kg 4 EGF-ld 17 days Equimolar 24 Hr Day6, 17 (n = 6 M; n = 7 F) post 5 EGF-ld 17 days Equimolar 24 Hr Day 6, 17(n = 7 M; n = 6 F) post

TABLE 6 GGF2 higher dose compared with EGF-ld and EGF-Ig Dosed for 20days starting day 7 after LAD. 10 day washout. ECHO Time In-Life DosingPoints Group Treatment Duration Dose Interval† (post-op) 1 N/A: Age 30days NA NA ‡Day 1, (n = 5 M; Matched post 12, 22, n = 5 F) Naïve primary& 32 controls ECHO 2 Control 38 days Vehicle 24 Hr *Day 7, 18, (n = 6 M;(Vehicle) post-op only 28, & 38 n = 6 F) 3 GGF-2 38 days 0.625 24 Hr*Day 7, 18, (n = 6 M; post-op mg/kg 28, & 38 n = 6 F) 4 GGF-2 38 days3.25 24 Hr *Day 7, 18, (n = 6 M; post-op mg/kg 28, & 38 n = 7 F) 5EGF-ld 38 days Equimolar 24 Hr *Day 7, 18, (n = 7 M; post-op 28, & 38 n= 6 F) 6 EGF-Ig 38 days Equimolar 24 Hr *Day 7, 18, (n = 7 M; post-op28, & 38 n = 6 F)

TABLE 7 GGF2 Dose frequency ECHO Time In-Life Dosing Points GroupTreatment Duration Dose Interval† (post-op) 1 N/A: Age 30 days NA NA‡Day 1, 12, (n = 5 M; Matched post 22, & 32 n = 5 F) Naïve primarycontrols ECHO 2 Control 38 days Vehicle 24 Hr *Day 7, 18, (n = 6 M;(Vehicle) post-op only 28, & 38 n = 6 F) 3 GGF-2 38 days 3.25 24 Hr *Day7, 18, (n = 6 M; post-op mg/kg 28, & 38 n = 6 F) 4 GGF-2 38 days 3.25 48Hr *Day 7, 18, (n = 6 M; post-op mg/kg 28, & 38 n = 7 F) 5 GGF-2 38 days3.25 96 Hr *Day 7, 18, (n = 7 M; post-op mg/kg 28, & 38 n = 6 F) TA1-Test Article 1; M = males; F = females.

TABLE 8 GGF2 with and without BSA ECHO Time In-Life Dosing Points GroupTreatment Duration Dose Interval† (post-op) 1 N/A: Age 17 days NA NA Day6 (n = 5 M; n = 5 F) Matched post-op and 17 Naive controls 2 Control 17days Vehicle 24 Hr Day 6 (n = 6 M; n = 6 F) (Vehicle) post only and 17 3GGF-2 + 17 days 3.25 24 Hr Day 6 (n = 6 M; n = 6 F) BSA post mg/kg and17 4 GGF-2 17 days 3.25 24 Hr Day 6 (n = 6 M; n = 7 F) without postmg/kg and 17 BSA

Test And Control Article Administration

Route of Administration

The test and control articles were administered by intravenousinjection. Animals assigned to Group 1 were not treated with vehicle orTest Articles; these animals served as age matched controls withouttreatment. Frequency of administration, duration, and dose were asdescribed in the Tables 5-8. The dose volume was approximately 1 ml perkg.

Test Article Administration

The test and control articles were administered via the tail vein.Individual doses were based on the most recent body weights. The dosewas administered by bolus injection, unless otherwise indicated by theSponsor.

Preparation of Test System

Surgical Procedure—Left Anterior Descending Artery Ligation

The surgical procedures were performed at Charles River Laboratories asdescribed in Charles River Laboratories Surgical Capabilities ReferencePaper, Vol. 13, No. 1, 2005. Briefly, a cranio-caudal incision is madein the chest, slightly to the left of the sternum, through skin and thepectoral muscles. The third and forth ribs are transected, and theintercostals muscles are blunt dissected. The thoracic cavity is rapidlyentered, and the pericardium completely opened. The heart isexteriorized through the incision. The pulmonary cone and left auricleare identified. A small curved needle is used to pass a piece of 5-0silk suture under the left anterior descending coronary artery. Theligature is tied, and the heart is replaced into the thorax. The air inthe thoracic cavity is gently squeezed out while the thoracic wall andskin incision is closed. The animal is resuscitated using positivepressure ventilation and placed in an oxygen rich environment.

Post-Operative Recovery

Short term post-operative monitoring and administration of appropriateanalgesics were performed by Charles River Laboratories as described inCharles River Laboratories Surgical Capabilities Reference Paper, Vol.13, No. 1, 2005.

Long term post-operative monitoring was conducted to assess the animalsfor signs of pain or infection. Daily incision site observationscontinued for 7 days post receipt of animals. Supplemental painmanagement and antimicrobial therapy were administered as necessitated.

TABLE 9 SCHEDULED MEDICATIONS AND DOSAGES INTERVAL, DOSE, AND ROUTE DAY32/38* DAILY DAY 1/7* DAY 12/18* DAY 22/28* ECHO & DRUG POSTSURGERY ECHOECHO ECHO Necropsy Isoflurane — To effect, To effect, To effect, Toeffect, inhalation inhalation inhalation inhalation Buprenorphine 0.01mg/kg, I.M. — — — — (only as needed) *ECHO procedure Day defined byanimal Group assignment as indicated below.

Antemortem Study Evaluations

Cageside Observations

All animals were observed at least twice a day for morbidity, mortality,injury, and availability of food and water. Any animals in poor healthwere identified for further monitoring and possible euthanasia.

Body Weights

Body weights were measured and recorded at least once prior torandomization and weekly during the study.

Food Consumption

Food consumption was not measured, but inappetence was documented.

Echocardiographic Examinations

Echocardiographic examinations were conducted on all animals assigned toGroup 1 on Day 1, 12, 22 and Day 32 post receipt (Day 0).Echocardiographic examinations were conducted on all animals assigned toGroup 2-5 on Day 7, 18, 28 and Day 38 post-surgical procedure conductedat Charles River Laboratories (Day 0).

For the echocardiographic examination, each animal was anesthetizedaccording to Table 5 and its hair clipped from the thorax. Coupling gelwas applied to the echocardiographic transducer and image obtained tomeasure cardiac function at multiple levels. Images were obtained foreach animal in short axis view (at mid-papillary level, or otherdepending on location of observed infarct area by echocardiography).

Echocardiographic Parameters

ECHO images were taken at the mid-papillary muscle level, or otherdepending on location of observed infarct area by echocardiography, ofthe left ventricle. M-mode and 2-D images were recorded and stored on CDand/or MOD. Measurement parameters obtained with ECHO include:Intraventricular Septal Wall Thickness (diastole); units=cm;Intraventricular Septal Wall Thickness (systole); units=cm; LeftVentricular Internal Dimension (diastole); units=cm; Left VentricularInternal Dimension (systole); units=cm; Left Ventricular Papillary WallThickness (diastole); units=cm; Left Ventricular Papillary WallThickness (systole); units=cm; End Diastolic Volume; units=mL; EndSystolic Volume; units=mL; Ejection Fraction; reported as a percentage;Stroke Volume; units=ml; and Percent Fractional Shortening; reported asa percentage

Euthanasia

Moribundity

Any moribund animals, as defined by a Testing Facility StandardOperating Procedure, were euthanized for humane reasons. All animalseuthanized in extremis or found dead were subjected to a routinenecropsy.

Method of Euthanasia

Euthanasia was performed by saturated potassium chloride injection intothe vena cava followed by an approved method to ensure death, e.g.exsanguination.

Final Disposition

All surviving animals placed on study were euthanized at their schedulednecropsy or, if necessary, euthanized in extremis.

RESULTS

Study 1—Treatment of rats with GGF2 at 0.625 mg/kg iv qday resulted insignificant improvement of cardiac function as shown here by changes inEjection Fraction and Fractional Shortening. EGF-1d fragment did notresult in the same degree of improvement. See Table 5.

Study 2—Treatment of rats with GGF2 at 0.625 and 3.25 mg/kg iv qdayresulted in significant improvement of cardiac function as shown here bychanges in Ejection Fraction and Fractional Shortening. Significantimprovements were also seen in end systolic and diastolic volumes duringthe treatment period. See Table 6.

Study 3 Results—Treatment of rats with GGF2 3.25 mg/kg iv q24, 48 or 96hours resulted in significant improvement of cardiac function as shownhere by changes in Ejection Fraction and Fractional Shortening.Significant improvements were also seen in end systolic and diastolicvolumes during the treatment period. See Table 7.

Previous reports (Liu et al) have shown that a carrier protein such asBSA is required for optimal neuregulin stability and activity. GGF2 hasdemonstrated stability without carriers such as BSA. This experiment wasdesigned to test whether GGF2 is stable and active in a therapeuticregimen without BSA. After 10 days of treatment, both the BSA andnon-BSA containing GGF2 formulations resulted in improvements inejection fraction compared with vehicle controls similar to those seenin previous studies. It is, therefore, evident from this study that BSAor other carrier protein is not required in GGF2 formulations for thetreatment of CHF. See Table 8.

TABLE 10 Pathology findings Sciatic Injection Nerve Sheath site/Hyperplasia Mammary Skin Cardiac Dosing (NSH) NSH changes effects Dailys.c. ++ ++ ++ + Daily i.v. + + + +/− 48 hour interval i.v. +/− − − +/−−96 hour interval i.v. − − − − ++ frequently present; + present; +/−occasionaly observed, − rare or not observed

As shown in Table 10, intermittent dosing of GGF2 reduces side effectsassociated with supranormal levels of exogenously administered GGF2. Thepresent inventors have discovered that this finding holds trueirrespective of whether the GGF2 is administered intravenously orsubcutaneously.

The hyperplasia and cardiac effects are sometimes seen with every otherday dosing. We have not seen with less frequent dosing.

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. All publications and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each independent publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

1. A method for treating or preventing heart failure in a mammal, saidmethod comprising: providing a peptide comprising an epidermal growthfactor-like (EGF-like) domain ; administering a therapeuticallyeffective amount of said peptide to a mammal at an interval of at least48 hours, wherein said therapeutically effective amount is effective totreat or prevent heart failure in said mammal.
 2. The method of claim 1,wherein said administering is performed every 48 hours.
 3. The method ofclaim 1, wherein said administering is performed every 96 hours.
 4. Themethod of claim 1, wherein said administration is performed on a regimenselected from the group consisting of every: four days, week, 10 days,14 days, month, two months, three months or four months.
 5. The methodof claim 1, wherein said mammal is a human.
 6. The method of claim 1,wherein said peptide is recombinant human GGF2.
 7. The method of claimwherein the peptide is: SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ.


8. The method of claim wherein the peptide is:SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQ NYVMASFYKAEELY.


9. The method of claim 1, wherein said peptide is encoded by theneuregulin (NRG)-1 gene, the neuregulin (NRG)-2 gene, the neuregulin(NRG)-3 gene, or the neuregulin (NRG)-4 gene.